1. Field of the Invention
The present invention relates to antibacterial peptides and antibacterial agents containing such peptides as an effective ingredient.
2. Description of the Related Art
An intention of keeping our living environmnent dean and comfortable has been emphasized by sensational prevalence of pathogenic Escherichia coli O-157, etc. On the other hand, drug resistant microbes such as MRSA are developed as a result of excessive use of antibiotics, which would throw a shadow of feature uneasiness.
Various kinds of antibacterial agents including antibiotics have been provided and used not only for medical purposes but as a variety of so-called antibacterial goods.
Antibacterial peptides have been getting popular in the course of developing antibacterial agents.
The above mentioned peptides are present in semen or blood serum of mamals and is considered as one of the most noteworthy antibacterial ingredients because of a wider antibacterial spectrum and high safety, as well as infrequent appearance of drug resistant microbe by use thereof.
Existence of an antibacterial peptide derived from body fluid of insects has been known as such peptides. Further, it has been known that various kinds of antibacterial peptides are derived in body fluid of insects when bacteria or different blood corpuscles are inoculated to insects or stimulation is given to a body surface thereof by wounding, etc.
For example, an antibacterial active peptide has been identified, which is derived from body fluid of a larval Zophobas atratus, an insect of Tenebriodae (see, J. Biol. Chem., vol. 266, pp. 24,524 to 24,525, 1991).
Further, physical and chemical properties of an antibacterial peptide named as Coleoptericin have been investigated.
On the other hand, an antibacterial peptide of cecropin group which is derived from body liquid of larval Hyalophora cecropia, an insect of Lepidoptera, itself and physical and chemical properties thereof have been found and investigated.
Antibacterial peptides derived from these insects have a wide antibacterial spectrum and accordingly are considered to bear important relation to biological defense of such insects which are lack of antibody forming ability.
One of problems associated with these antibacterial peptides includes antigenicity. Each of antibacterial peptides has not so low molecular weight and can be antigen in the body when they are vascularly administered as an antibacterial agent.
Because of the wide but somewhat biased antibacterial spectrum, there are not necessarily provided antibacterial peptides which are sufficiently effective to both Gram-positive and Gram-negative bacteria.
Further, there is no gainsaying possible development of some cytotoxicity when these insect-derived antibacterial peptides are given to mammals.
Accordingly, it is an object of the present invention to provide antibacterial measure by use of antibacterial peptides which overcome conventional problems as described above.
In order to solve conventional problems, structure and functions of an antibacterial peptide derived from insects of Lepidoptera such as Scarabaeidae, Tenebriodae, etc., especially Oryctes rhinoceros which is an insect of Eophileurus chinensis of Scarabaeidae have been investigated as an insect-derived peptide by the present inventor.
It is known that an antibacterial peptide is induced in body liquid of larva Oryctes rhinoceros when stimulated by, for example, wounding. Using cDNA of larva Oryctes rhinoceros, its nucleotide sequence encoding an antibacterial peptide (hereinafter referred to as natural antibacterial peptide gene) was identified.
Then, amino acid sequence of an antibacterial peptide derived from Oryctes rhinoceros (hereinafter referred to as natural antibacterial peptide unless otherwise noted) was estimated from the nucleotide sequence thus identified. Development of novel antibacterial peptides has been done on the basis of information of such amino acid sequence.
[1] Nucleotide sequence identification of natural antibacterial peptide cDNA
The nucleotide sequence of natural antibacterial peptide cDNA was identified by a conventionally known method as in the following.
(1) The natural antibacterial peptide was extracted from larva Oryctes rhinoceros and purified to identify N-terminal amino acid sequence thereof Based on the amino acid sequence, a degenerate primer was synthesized. In addition, another degenerate primer was also synthesized based on the conserved amino acid sequence of the C-terminal region of the insect antibacterial peptide family designated defensive.
(2) Using these oligonucleotides as primers, a portion which would code the antibacterial peptide cDNA, was subjected to manipulation of gene amplification (PCR method).
(3) The nucleotide sequence of PCR product was determined.
In order to identify N-terminus of the natural antibacterial peptide in the above mentioned process (1), separation and purification of the natural antibacterial peptide from larva Oryctes rhinoceros were carried out as in the following.
Ten of the third instar larvae of Oryctes rhinoceros were kept on ice for several minutes to slow down their movement and wounded by piercing a hypodermic needle. The thus wounded larvae were kept in a breeding box with leaf mold for 24 to 48 hours at a temperature of 25xc2x0 C.
After cutting legs of wounded larva Oryctes rhinoceros, drops of their body liquid was squeezed and collected in a tube on ice by pressing abdomen thereof. As a result, about 1.5 ml of body liquid was obtained from each larva.
After the above mentioned body liquid was subjected to centrifugal separation (39,000xc3x97 g) for 50 minutes, a blood corpuscle component was removed and the supernatant thus obtained was kept at a temperature ofxe2x88x9220xc2x0 C. Then, 15 ml of the supernatant was applied to Sep-Pak C18-cartridge (available from Waters Associates Co., Ltd.) which had been equilibrated with a solvent A containing 20% of acetonitrile, followed by washing the column with the solvent A, to elute an antibacterially active substance (active fraction) with a 20%-acetonitrile/0.05%-trifluoroacetic acid solution.
The active fraction was freeze-dried to remove acetonitrile and form a dried powder, which was dissolved in a 0.05%-trifluoroacetic acid solution to apply to Resource RPC Column (1 ml; available from Pharmacia Co., Ltd.) which had been equilibrated with a 0.05%-heptafluorobutytir acid (HFBA) solution.
The column was washed with the 5%-HFBA solution, followed by gradient elution under a condition of 0% to 20%-acetonitrile/0.05%-trifluoroacetic acid solution for 1.25 minutes; similarly a 20% to 40%-solution for 10 minutes and a 40% to 100%-solution for 1.25 minutes. It was observed that an 38 to 40%-acetonitrile fraction is antibacterially active.
The antibacterial activity was observed as in the following.
To 0.99 ml of a bouillon medium prepared by dissolving 1 g of beef extract (available from Difco Co., Ltd), 2 g of Bacto-peptone (available from Difco Co., Ltd.) and 1 g of NaCl in 200 ml of water, 10 xcexcl of bacteria suspension prepared by culturing Staphylococcus aureus in bouillon medium overnight was added and cultured up to period of logarithmic multiplication.
To bouillon-agar medium prepared by adding 1.5% by weight of agar to the bouillon medium, 200 xcexcl of the thus obtained culture solution was added and poured to a laboratory dish to gelate it. Then, a well of 2 mm in diameter was formed on the gelled bouillon-agar medium by means of a gel puncher (available from Parmacia Co., Ltd.). Five xcexcl of 38 to 40%-acetonitrile fraction (sample) dissolved in water was poured into the well and cultured overnight at a temperature of 37xc2x0 C. to observe a hollow formed thereon.
A procedure described above was repeated similarly.
The active fraction was further freeze-dried to remove acetonitrile and form a dried powder, which was dissolved in the solvent A to apply to a column (3.2xc3x9730 mm, xcexc RPC C2/C18 PC3.2/3 column, available from Pharmacia Co., Ltd.). After the column was washed by the solvent A, elution was carried out under a mild gradient condition of 20% to 40%-acetonitrile/0.05%-trifluoroacetic acid for 40 minutes. It was observed that an 23 to 24%-acetonitrile fraction is antibacterially active.
The thus obtained fraction of natural antibacterial peptide was applied to a reversed column (2.1xc3x97100 mm, xcexc RPC C2/C18 SC2.{fraction (1/10)}, available from Pharmacia Co., Ltd.) which had been equilibrated with the solvent A. The reversed column was connected to SMART system (available from Pharmacia Co., Ltd.) and washed by the solvent A, followed by elution under a mild gradient condition 0% to 20%-acetonitrile/0.05%-trifluoroacetic acid for 5 minutes; 20% to 40% for 40 minutes and 40% to 100% for 5 minutes.
The thus obtained eluent was fractionated to each 200 xcexcl-fraction, which was then freeze-dried to observe the antibacterial activity one after the other. The antibacterial activity was eluted as a single peak compatible with that of protein as shown in FIG. 1. Mass spectrometry (MALDI-TOF-MS) of the antibacterially active fraction showed a single molecular weight of 4,489 (M+Na) as shown in FIG. 2.
The thus obtained fraction, which was considered as the natural antibacterial peptide, was subjected to Edman degradation to determine the amino acid sequence. It was found that the sequence of 15 PTH-amino acid residues (sequence No. 1) which would correspond to a N-terminus of the natural antibacterial peptide is as in the following: 
The antibacterial activity of the present natural antibacterial peptide was determined by changing an amount of protein from 0 to 0.8 in 50 xcexcl of test solution. The result is shown in FIG. 3, from which it is confirmed that the antibacterial activity of this peptide is clearly effective to Staphylococcus aureus. Further, the natural antibacterial peptide was subjected to a heat treatment at a temperature of 100xc2x0 C. for 5 to 10 minutes, thereby the initial antibacterial activity being kept.
A degenerate oligonucleotide primer was synthesized based on the amino acid sequence of the N-terminal region of natural antibacterial peptide following the procedure described above (I).
On the other hand, another degenerate primer was synthesized based on the amino acid sequence of the conserved C-terminal region of the closely related antibacterial peptide family.
The first strand cDNA was prepared using a restricted enzyme Not I site-added primer with fat body mRNA of Escherichia coli-infected third instar larva Oryctes rhinoceros as a template, to which PCR-gene amplification was carried out using the above mentioned PCR primer. As a result, an amplified gene product of about 120 bp in length was obtained.
Nucleotide sequence of the amplified gene product was determined, while a primer was further prepared according to the nucleotide sequence and subjected to the PCR-amplification between a complementary primer to the restricted enzyme Not I site which was used to prepare the first strand cDNA. As a result, nucleotide sequence of the amplified gene product at the 3xe2x80x2-terminal side was elucidated.
Further, the first strand cDNA was prepared using the primer based on the base sequence thus identified with fat body mRNA of Escherchia coli-infected third instar larva Oryctes rhinoceros as a template. Then, poly C sequence was added to the cDNA at the 5xe2x80x2-terminal, which was used as a template for a primer having poly G sequence. Using thus prepared primer and the above mentioned primer, which was prepared according to the identified nucleotide sequence, as PCR primers, the PCR amplification was conducted. Nucleotide sequence of the amplified gene product at the 5xe2x80x2-side was determined.
Nucleotide sequence information thus clarified one after the other were put together to confirm cDNA nucleotide sequence which is capable of coding the natural antibacterial peptide derived from Oryctes rhinoceros (See, Sequence No. 2). The amino acid sequence estimated from this nucleotide sequence is also shown in Sequence No. 2.
DNA having the Sequence No. 2 was integrated into a congenic vector (pCR 2-1; available from Invitrogen Co., Ltd.) to transform Escherichia coli JM 109. The transformant has been deposited to the National Institute of Bioscience and Human-Technology (NIBH) as pCROAD (Dpodition No. FERM P-16,800).
As a result of comparison of the estimated amino acid sequence represented by the Sequence No. 2 and that of the natural antibacterial peptide at the N-terminus in the Sequence No. 1, it was confirmed that amino acid residues from the first Met to the 36th Arg are extracellularly secreting signal peptide and propeptide, while those residues from the 37th Leu to Arg just prior to the last codon are maturated natural antibacterial peptide which actually exhibits antibacterial activity.
The natural antibacterial peptide, whose amino acid sequence has been confirmed as described above, comprises 43 amino acid residues and exhibits high antibacterial activity.
[2] Preparation of novel peptide
Size of the above mentioned peptide comprising 43 amino acid residues, however, is large enough to function as an immunogen in an in vivo immune system. That is, it is undesirable that the natural antibacterial peptide might activate the immune system when such a peptide is given to an organism.
Further, the natural antibacterial peptide has an antibacterial spectrum only against Gram-positive bacteria but exhibits little activity against Gram-negative bacteria.
Accordingly, the present inventor has investigated the amino acid sequence of the natural antibacterial peptide in detail and found that residues from the 62nd Ala to the 69th Ala of the above mentioned amino acid sequence are in charge of the antibacterial activity. It is thus considered that activation of the host immune system caused by the natural antibacterial peptide can be reduced when an active center portion thereof is used as an antibacterial agent.
It is also considered that a change in amino acid sequence around the active center portion may improve in an antibacterial spectrum not only to Gram-positive but Gram-negative bacteria.
It is finally found that certain peptide which comprises specific amino acid sequence having amidated C-terminus as a core portion has high antibacterial activity against both Gram-positive and Gram-negative bacteria, although molecular size thereof is relatively small. On the basis of this information, the present invention has been completed.
The present invention provides antibacterial peptides represented by the following formula:
X-Ala-Ile-Arg-Lys-Arg-NH2xe2x80x83xe2x80x83(1)
wherein X is peptide in which one or more than two amino acid residues are linked by peptide linkage, and Arg-NH2 means that a carboxyl group of Arg is amidated.
There are also provided antibacterial agents containing the antibacterial peptide as an effective ingredient and, in particular, edible antibacterial agents which can be taken in digestive tracts.